Molecular characterization and sensitivity to demethylation inhibitor fungicides of Aspergillus fumigatus from orange-based compost

Aspergillus fumigatus, the causal agent of human aspergilloses, is known to be non-pathogenic in plants. It is present as saprophyte in different types of organic matter and develops rapidly during the high-temperature phase of the composting process. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides and resistant isolates have been recently reported. The present study aims to estimate the abundance, genetic diversity and DMI sensitivity of A. fumigatus during the composting process of orange fruits. Composting of orange fruits resulted in a 100-fold increase in A. fumigatus frequency already after 1 week, demonstrating that the degradation of orange fruits favoured the growth of A. fumigatus in compost. Most of A. fumigatus isolates belonged to mating type 2, including those initially isolated from the orange peel, whereas mating type 1 evolved towards the end of the composting process. None of the A. fumigatus isolates expressed simultaneously both mating types. The 52 investigated isolates exhibited moderate SSR polymorphisms by formation of one major (47 isolates) and one minor cluster (5 isolates). The latter included mating type 1 isolates from the last sampling and the DMI-resistant reference strains. Only few isolates showed cyp51A polymorphisms but were sensitive to DMIs as all the other isolates. None of the A. fumigatus isolates owned any of the mutations associated with DMI resistance. This study documents a high reproduction rate of A. fumigatus during the composting process of orange fruits, requesting specific safety precautions in compost handling. Furthermore, azole residue concentrations in orange-based compost were not sufficient to select A. fumigatus resistant genotypes.烟曲霉（Aspergillus fumigatus）是人类曲霉病的致病原，已知其对植物无致病性。该菌以腐生菌的形式存在于多种有机质中，并在堆肥过程的高温阶段快速增殖。曲霉病的临床治疗常使用脱甲基化抑制剂（DMI）类杀菌剂，近期已有该菌耐药分离株的相关报道。本研究旨在评估柑橘果实堆肥过程中烟曲霉的丰度、遗传多样性以及对DMI类杀菌剂的敏感性。 柑橘果实堆肥仅1周后，烟曲霉的检出频率便提升了100倍，表明柑橘果实的降解可促进堆肥中烟曲霉的生长。绝大多数烟曲霉分离株属于交配型2，包括最初从柑橘果皮中分离得到的菌株；而交配型1仅在堆肥过程末期被检出。未发现同时携带两种交配型的烟曲霉分离株。 本次研究的52株分离株呈现出中等程度的简单序列重复（SSR）多态性，可分为一个主要类群（47株）与一个次要类群（5株）。次要类群包含最后一次采样得到的交配型1分离株以及DMI耐药参考菌株。仅少数分离株存在细胞色素P450 51A（cyp51A）基因多态性，但与其余所有菌株一样，它们对DMI类杀菌剂仍保持敏感性。未发现任何一株烟曲霉分离株携带与DMI耐药性相关的突变位点。 本研究证实烟曲霉在柑橘果实堆肥过程中具有极高的繁殖速率，提示在堆肥处理过程中需采取针对性的安全防护措施。此外，柑橘基堆肥中的唑类杀菌剂残留浓度不足以筛选出烟曲霉耐药基因型。